The proposed research is a continuation and extension of previous investigations of fundamental biological mechanisms regulating the replication and transfer of bacterial plasmids. It is, in effect, a multifaceted study involving 1. investigation of the structural and functional basis of the control of plasmid replication, incompatibility (i.e., replication inhibition), and copy number, 2. isolation and characterization of plasmid DNA sequences that serve as replication origins and study of possible interrelationships between the primary nucleotide sequence and the replication-assciated properties of the plasmid, 3. study of the biological role and incompatibility relationships of multiple regions of replication activity present on naturally occurring plasids, 4. investigation of species-specific factors that determine the ability of a plasmid to replicate in a particular related or unrelated species,5. investigation of the mechanism of plasmid mobilization, including the posssble role of transposable DNA sequences in plasmid mobilization, 6. isolation and characterization of plasmid DNA regions specifying functions for interbacterial transfer of extrachromosomal DNA and 7. study of gene products involved in the replication and transfer of plasmids. The proposed experiments are made possible by recently developed methods for cleaving and joining plasmid DNA molecules at specific sites using restriction endonucleases and DNA repair enzymes, and for introducing plasmid DNA into various bacterial species by transformation and/or protoplast fusion. In addition, procedures that enable the rapid determination of the primary nucleotide sequence of DNA segments, and improved methods for the de novo synthesis of short chains of polydeoxyribonucleotides now make possible a level of investigation of plasmid replication and transfer that was not possible previously.